EP3691474A1 - Dietary fiber composition for protecting bioactive compounds during the physiological digestion process - Google Patents

Dietary fiber composition for protecting bioactive compounds during the physiological digestion process

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Publication number
EP3691474A1
EP3691474A1 EP18773170.8A EP18773170A EP3691474A1 EP 3691474 A1 EP3691474 A1 EP 3691474A1 EP 18773170 A EP18773170 A EP 18773170A EP 3691474 A1 EP3691474 A1 EP 3691474A1
Authority
EP
European Patent Office
Prior art keywords
composition
extract
weight
amount
polar nature
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP18773170.8A
Other languages
German (de)
French (fr)
Inventor
María Dolores DEL CASTILLO BILBAO
Antonio Manuel INAREJOS GARCÍA
Jean-Marie RAYMOND
Marin PRODANOV PRODANOV
Miriam GRANADO GARCÍA
Daniel GONZALEZ HEDSTRÖM
Paula ALMODOVAR HORTELANO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmactive Biotech Products SL
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
Original Assignee
Pharmactive Biotech Products SL
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Autonoma de Madrid
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmactive Biotech Products SL, Consejo Superior de Investigaciones Cientificas CSIC, Universidad Autonoma de Madrid filed Critical Pharmactive Biotech Products SL
Publication of EP3691474A1 publication Critical patent/EP3691474A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/238—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seeds, e.g. locust bean gum or guar gum
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a composition
  • a composition comprising galactomannans, fructooligosaccharides (FOS) and bioactive compounds of polar nature, mainly, phenols and polyphenols from carob ⁇ Ceratonia siliqua L) pod extract.
  • the adequate ratio of galactomannans (soluble fibers) and fructooligosaccharides (insoluble fibers) protects the bioactive compounds of polar nature against degradation during digestion, increasing their bioavailability thereof in the organism.
  • the present invention refers to the field of administration of bioactive compounds.
  • International patent application WO2012/044242 relates to a food product containing viscous fibres (including carob gum) between 4 and 35% by dry weight. Additionally, one or more components selected from the group of indigestible carbohydrates (for example fructooligosaccharides), polyphenols, fatty acids and probiotics can be added.
  • the claimed food product may improve glucose tolerance/insulin sensitivity already in the late post-prandial phase following acute meal.
  • Patent application US2003/0059458 discloses a composition of water insoluble carob fibre in an amount of about 10 to about 90% by weight and an encapsulating material of at least one water soluble dietary fiber in an amount of about 10 to about 90%, being the water soluble dietary fibre a natural or a chemically modified polysaccharide.
  • compositions that specifically intend to protect bioactive compounds during digestion mainly by using polysaccharide carriers, contribute to the increase of blood glucose content of diabetic subjects. Accordingly, there is a need for providing compositions not only rich in bioactive compounds of polar nature but also that have increased bioavailability thereof and, at the same time, that reduce blood glucose content after consumption.
  • the inventors of the present invention have discovered that when a composition comprises galactomannan (soluble fibers), hereinafter GLMN, in an amount of 36 to 40% by weight, based on the total weight of the composition and fructoligosaccharides, hereinafter FOS, (non-soluble fibers) in an amount of 2 to 4% by weight, based on the total weight of the composition, the extract of polar nature present in said composition are, surprisingly, protected from being digested in the stomach (see example 2), avoiding the almost complete loss of the bioactive compounds contained in the extract during the digestion process, and resulting in a higher bioavailability of these compounds after digestion. As a consequence, the amount of bioactive compounds to be administered can be decreased because there is a low loss thereof during digestion.
  • GLMN galactomannan
  • FOS fructoligosaccharides
  • the inventors have also discovered that, not only the bioactive compounds included in the extract of polar nature are present after digestion, but also soluble and insoluble fibre, and therefore, the composition of the present invention may play a role to prevent from risk factors that commonly cluster together (like dyslipidaemia, hypertension and hyperglycaemia) and have been termed as metabolic syndrome.
  • ATP III National Cholesterol Education Program's Adult Treatment Panel III report
  • CVD cardiovascular disease
  • the pathogenesis of the metabolic syndrome is not known, but there seem to be three potential etiological categories: obesity and disorders of adipose tissue, insulin resistance and a number of independent factors that mediate specific components of the metabolic syndrome (Beilby, J. 2004, Clin Biochem Rev. 25(3): 195-198).
  • these compounds are useful for the manufacture of a composition of soluble and insoluble fibres for the administration of several functional molecules and/or for the development of nutraceuticals more effective at even lower doses.
  • an adequate composition of soluble and insoluble fibres is presented as a natural "encapsulating carrier" to protect bioactive compounds of polar nature, such as phenolic compounds, from digestion process and therefore to improve their bioavailability and beneficial effect on the organism.
  • composition of the invention relates to a composition, hereinafter "composition of the invention", comprising GLMN in an amount of from 36 to 40% by weight, based on the total weight of the composition, a FOS in an amount of from 2 to 4% by weight, based on the total weight of the composition, and at least one extract of polar nature.
  • composition refers to any substance comprising any combination of three or more molecules, wherein said molecules are, at least, GLMN, a FOS and an extract of polar nature containing bioactive compounds such as phenolic compounds.
  • galactomannan refers to a polysaccharide consisting of a mannose backbone with galactose side groups, more specifically, a (1 -4)-linked ⁇ -D- mannopyranose backbone with branchpoints from their 6-positions linked to a-D- galactose, i.e. 1 -6-linked a-D-galactopyranose, as represented in Figure 1.
  • Galactomannan can be isolated from multiple sources.
  • locust bean Ceatonia siliqua
  • guar Cyamopsis tetragonoloba
  • tara Croposa Kuntze
  • fenugreek Trigonella foenum- graecum L
  • galactomannans include, without limiting to, fenugreek gum (mannose:galactose -1 :1 ), guar gum (mannose:galactose ⁇ 2:1 ), tara gum (mannose:galactose -3:1 ) and locus bean gum or carob gum (mannose:galactose -3:1 ).
  • fenugreek gum mannose:galactose -1 :1
  • guar gum mannose:galactose ⁇ 2:1
  • tara gum mannose:galactose -3:1
  • locus bean gum or carob gum mannose:galactose -3:1
  • Any galactomannan can be used in the composition of the invention.
  • the galactomannan comprises a mannose:galactose ratio of about 3:1 from carob (Ceratonia siliqua L) gum.
  • the source of galactomannan is the endosperm of carob seed (Ceratonia siliqua L) ground with an amount of galactomannan no less than 75% (dry weight) (see Figure 2).
  • the amount of galactomannan in the final fiber complex (composition) of the present invention is 36% by weight based on the total weight of the composition.
  • the fructooligosaccharide refers to oligofructose produced by partial enzymatic hydrolysis of chicory inulin.
  • Oligofructose consist of fructose units linked by ⁇ (2-1 ) linkages. The degree of polymerization chiefly ranges between 2 and 8.
  • the FOS comprises no less than 90% by weight of oligofructose (dry matter).
  • the amount of fructooligosacharide is 3% by weight based on the total weight of the composition.
  • the amount of GLMN is 36% by weight based on the total weight of the composition and/or the amount of FOS is 3% by weight based on the total weight of the composition.
  • the present invention is based on the fact that the presence of GLMN and FOS in a particular amount protects the bioactive compounds contained in the extract of polar nature present in a composition from digestion process.
  • extract refers to a substance obtained from plants by extracting a part of a raw material, often by using a solvent such as ethanol, water or a combination of both.
  • bioactive compounds refers to the ingredients or components which are biologically active, i.e. are responsible for conferring to the composition therapeutic properties, and these compounds are soluble in water or in mixtures of water and ethanol [not more than 20% of ethanol (v/v) in the final solvent mixture]
  • the presence of fibres in the final product protects the bioactive compounds contained in the extract of polar nature during the digestion process, chiefly phenolic compounds.
  • Any natural extract of polar nature can be used in the context of the present invention.
  • Various extracts and plant powders are incorporated into the composition of the invention depending on the desired properties according to the end use of said compositions.
  • extracts with therapeutic properties include, without being limited to, botanical extracts standardized to bioactive compounds, such as, one or more of those of Panax ginseng (red, Korean ginseng), Panax ginseng (white, Chinese ginseng), Rhodiola rosea (golden root), Panax quinquefolium (American ginseng), Eleutherococcus senticosus (Siberian ginseng), Cynara scolymus (artichoke), Uncaria tomentosa (Cat's claw), Lepidium meyenii (maca, Peruvian ginseng), Paullinia cupana (guarana), Croton lechleri (Sangre de Grado), Whitania somnifera (ashwagandha, Indian ginseng), Panax japonicus (Japanese ginseng), Panax vietnamensis (Vietnamese ginseng), Panax trifolius, Panax pseudoginseng, Panax
  • the extract of polar nature is derived from carob, particularly, from carob pods.
  • the extracts used as bioactive compounds in the composition of the invention can include multiple antioxidant compounds such as phenols, polyphenols, secoiridoids, peptides, carotenoids, enzymes, gluthatione, hormones, lipids, minerals, vitamins, saponines, steroids, etc.
  • antioxidant compounds such as phenols, polyphenols, secoiridoids, peptides, carotenoids, enzymes, gluthatione, hormones, lipids, minerals, vitamins, saponines, steroids, etc.
  • phenols include, but without being limited to, simple phenolic acids and derivatives, hydroxycinnamic acids and derivatives, flavonoids (flavones, isoflavones, flavanones, flavonols, anthocyanins), tannins (gallotannins and ellagitannins), and related components of polar nature.
  • the extract of polar nature included in the composition of the invention comprises hydrolysable tannins derived from gallic acid.
  • these compounds may represent between 50 to 90% by weight of the extract of polar nature, and more preferably 70% by weight.
  • the amount of the extract of polar nature in the composition of the invention can encompass a wide range of concentrations. Nevertheless, in a particular embodiment, the composition comprises an amount of the extract of polar nature from 30 to 49 % by weight based on the total weight of the composition, preferably 40% by weight.
  • the composition of the invention may be a pharmaceutical or a nutritional composition.
  • the pharmaceutical and the nutritional composition may be formulated, but not limited to, in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, microcapsule, powder, granule, ointment, solution, paste, suppository, injection, inhalant, gel, microsphere or aerosol.
  • the composition is formulated for oral administration.
  • composition of the invention comprises a prophylactically or therapeutically effective amount of at least one bioactive compound of polar nature.
  • prophylactically effective amount means that the bioactive compound contained in the composition is in sufficient quantity to exert its effect on the subject. The effect will depend on the kind of the bioactive compound used in the composition of the invention. Examples of bioactive compounds have been disclosed above.
  • compositions of the present invention may include a carrier.
  • a carrier may be a dietary suitable carrier or a pharmaceutically acceptable carrier, as long as it is compatible with the bioactive principle of the composition.
  • a dietary suitable carrier include, but are not limited to, dietary suitable excipients and diluents.
  • excipient relates to a substance that aids the absorption of any of the bioactive compound of the composition of the invention, stabilizes said bioactive compound or aids the preparation of the composition in the sense of giving consistency or contributing flavours that make it more enjoyable.
  • excipients may have the function of holding the components together, such as starches, sugars or celluloses, a sweetening function, colouring function, drug protection function, the function of filling a tablet capsule, sachets, softgels and related galenic forms. Therefore, the term "excipient" is defined as the material included in the galenic forms, is added to the bioactive compounds or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties of the pharmaceutical composition and its bioavailability.
  • the term "nutritional composition” relates to a food or a dietary supplement that regardless of providing nutrients to the subject has a beneficial effect on one or more health functions, so as to provide a better state of health and well-being. Accordingly, such nutritional composition may be destined for the prevention and/or treatment of a disease or for the reduction of disease risk factors, such as those related wit metabolic syndrome.
  • the present invention relates to the composition of the invention for use as a medicament.
  • the present composition can be used for treating and/or preventing a particular disease.
  • the invention when the bioactive compounds are coming from a carob extract, particularly, carob pod extract, the invention relates to a composition for use in the treatment and/or prevention of risk factors related to metabolic syndrome.
  • treatment relates to fight the effects of a disease or pathological condition of interest in a subject (preferably a mammal and, more preferably, a human) that includes: (i) inhibiting the disease or pathological condition, i.e. arresting its development;
  • prevention consists of preventing the onset of the disease, that is, preventing the development of disease or pathological condition from occurring in a subject (preferably a mammal and, more preferably, a human), particularly when said subject is predisposed to develop the pathological condition.
  • metabolic syndrome refers to the set of metabolic alterations that jointly increase the risk of diabetes and cardiovascular disease, including the combination of obesity, dyslipidaemia (for example, triglycerides and hypercholesterolemia) and hyperglycaemia.
  • the present invention relates to the composition for use in increasing the absorption of an extract of polar nature in a subject with the following composition: - galactomannans in an amount of from 36 to 40% by weight based on the total weight of the composition,
  • fructooligossacharides in an amount of from 2 to 4% by weight based on the total weight of the composition
  • the amount of galactomannan is 36% by weight based on the total weight of the composition and/or the amount of fructooligosascharide is 3% by weight based on the total weight of the composition.
  • the galactomannan comprises a mannose:galactose ratio of about 3:1.
  • the fructooligossacharides comprise no less than 90% by weight of oligofructose (%, dry matter).
  • the extract of polar nature comprises hydrolysable tannins derived from gallic acid.
  • the hydrolysable tannins derived from gallic acid represent between 50 to 90% by weight of the extract of polar nature, preferably 70% by weight.
  • the extract of polar nature is derived from carob, preferably from carob pods.
  • the extract of polar nature is in an amount of from 30 to 49% by weight based on the total weight of the composition, preferably 40% by weight.
  • the extract of polar nature is a mixture of one or more botanical extract different from carob extract. Examples of botanical extracts have been disclosed previously in the present disclosure.
  • the invention relates to a method for obtaining the composition of the invention, hereinafter "method of the invention", comprising mixing an extract of polar nature with (i) galactomannan in an amount of from 36 to 40% by weight based on the total weight of the composition and (ii) FOS in an amount of from 2 to 4% by weight based on the total weight of the composition.
  • the amount of galactomannan is 36% by weight based on the total weight of the composition and/or the amount of FOS is 3% by weight based on the total weight of the composition.
  • the galactomannan comprises a mannose:galactose ratio of about 3:1.
  • the galactomannan comes from carob seeds and/or the FOS comes from inulin.
  • the extract of polar nature is carob extract, particularly, carob seed extract or carob pod extract.
  • Figure 1 discloses a segment of galactomannans showing mannose backbone (below) with a branching galactose unit (top), wherein m>1 and n>1.
  • Figure 2 shows the chemical structure of carob galactomannans. It is a linear chain of 1 ,4- ⁇ - ⁇ mannopyranose units with 1 ,6-linked a-D galactopyranose units attached to every fifth mannose unit.
  • Figure 3 shows a manufacturing flow chart of the carob (Ceratonia siliqua L) pod extract.
  • Black line marks the basal blood flow. Values are normalized to 100% with their basal blood flow and represented as the meaniSEM.
  • the samples tested comprised carob extract as an extract of polar nature, galactomannans from locust bean gum (>75% galactomannans) and fructooligosaccharides (>90% purity).
  • Deseeded and chopped carob pods were obtained, ground and then extracted according to the flow chart ( Figure 3), mixed and packaged in the processing factory of Pharmactive (Madrid, Spain).
  • the carob kibbles were stored in a cool and dry place until analysis and extraction process.
  • Temperature control is a critical parameter for the extraction process. It should be limited up to 85°C in order to avoid the deterioration of some phenolic compounds. Organic solvents such as ethyl acetate, hexane, petroleum ether, acetone, methanol or the like are not suitable for carob pod extraction.
  • the crude extract is a dense liquid, with intense brown colour and characteristic aroma of carob (Ceratonia siliqua L). Once cooled to room temperature the extract is concentrated and dried, using different drying techniques.
  • the dry solid extract is then milled; the last step comprised of adjusting the particle size by milling and grinding the dry extract in a Hammer or blades mill with a strict temperature control to avoid bioactive compound degradation. Finally, the powder solid extract is sieved through a light sieve to obtain a maximum particle size of 240 ⁇ .
  • the obtained extract was characterized by HPLC, showing the following profile of components:
  • Table 1 Identified individual polar phenolic compounds in carob pod extract and their retention time (t R ), UV-VIS spectra, quasi-molecular [M-H] " and fragment ions, acquired in negative ionisation mode.
  • t R retention time
  • Tetragalloyl 223/272 786.9 635.0, 483.0, 330.8, 313.0, hexoside 271.0, 211.0, 168.9
  • Batch I and Batch II two different batches were obtained from carob pod extracts, called Batch I and Batch II, enriched in bioactive compounds of polar nature.
  • Batch I or Batch II are mixed with the soluble/insoluble fibres complex (galactomannans from carob seeds and FOS from inulin) in order to obtain the final product (composition) of the present invention.
  • the final amounts of the carob pods extract, galactomannans and FOS in the compositions are, respectively, 40, 36 and 3%, by weight, based on the total weight of the composition. Due to the homogeneity of the composition of the present invention, the results obtained with Batch I and Batch II were considered together as can be observed in Table 1 and also in Table 2a for the statistical analyses during the digestion process.
  • compositions obtained from two different batches of carob extracts denominated Batch I and Batch II together with GLMN and FOS was analysed before and after in vitro digestion for bioaccesibility of bioactive compounds: total polar phenols with antioxidant capacity, together with glycaemic sugars (Table 2a).
  • an in vitro digestion protocol comprises the following three phases: 1 ) cephalic or salivary phase, 2) gastric phase, and 3) intestinal or duodenal phase. All processes were carried out in one and the same tube.
  • an in vitro abiotic digestion was carried out, comprising three stages (oral, gastric and intestinal) as described by Hollebeeck et al., 2013. Briefly, the salivary step was performed with 3.9 units of ⁇ -amilase/mL of sample, at pH 6.9, 37°C, 5 min of incubation, and aerobic conditions.
  • the gastric step was performed with 71 .2 units of pepsin/mL of sample at pH 2, 37°C, 90 min of incubation, and anaerobic conditions while the abiotic duodenal step was run with a mixture of 9.2 mg of pancreatin and 55.2 mg of bile extract/mL of sample at pH 7, 37°C and 150 min, under anaerobic conditions.
  • the final mixture was centrifuged at 5000 rpm for 40 min, at 4°C. A soluble fraction was obtained by centrifugation which was freeze-dried. The resulting powder was stored at -20°C until analysis. This process is performed in duplicate, and the corresponding digested samples are analysed in triplicate.
  • Folin-Ciocalteu adapted to a micromethod format was the test selected for analysis of total polar phenolic compounds in the different samples, before and after the digestion process (Contine, Baccelini, Massantini & Anelli, 2008).
  • the reaction was initiated by mixing 10 ⁇ _ of sample with 150 ⁇ _ of Folin-Ciocalteu reagent. After incubation at room temperature during 3 min, 50 ⁇ _ of sodium bicarbonate solution were added. The kinetics of the reaction at 37 °C was followed for 120 min by measuring the absorbance at 735 nm once every minute. A gallic acid calibration curve was used for quantification. Results are expressed as percentage (% by dry weight). All measurements were performed in triplicate.
  • ABTS ' decolourisation assay was performed according to Oki, Nagai, Yoshinaga and Nishiba (2006).
  • An ABTS '+ aqueous solution was prepared by adding 140 mM potassium persulfate (44 ⁇ _) to a 7 mM ABTS '+ aqueous solution (2.5 ml_), and the mixture was then allowed to stand for 16 h at room temperature.
  • the working solution of the radical ABTS '+ was prepared by diluting the stock solution 1 :75 (v/v) in 5 mM sodium phosphate buffer pH 7.4 to obtain an absorbance value of 0.7 ⁇ 0.02 at 734 nm.
  • Insoluble and soluble (galactomannan) fibres were determined using respectively the "Total Dietary Fibre Assay Kit” and the “Galactomannan Kit” (Megazyme International Ireland, Ireland), based on an enzymatic-gravimetric method described in the manufacturer ' s instructions. Results were expressed as % (dry weight).
  • the capsules with the composition of the invention employed for digestion were assessed for their moisture content. For this, samples of about 1 g of the solid powder were heated at 1 10°C for 15 min and cooled in a desiccator to room temperature. The moisture content was determined by the gravimetric method according to the AOAC-925.10. The samples were weighted accurately ( ⁇ 1 g), and dried until constant weight in an oven at 105°C. Results were expressed as %.
  • Glucose, mannose and fructose were determined employing the Megazyme kit according to the manufacturer ' s instructions (Megazyme KMANGL, Ireland) adapted to micro-method format.
  • a Bio Tek Power Wave TM XS microplate reader Bio Tek Instruments, USA was used to measure the samples absorbance at 340 nm.
  • D- mannose, D-glucose, D-mannose and D-fructose contents in each sample were expressed as % of glucose, mannose and fructose (dry weight).
  • Capacity Ext+GLMN is the extract (average results of Batch I and Batch II) together with galactomannans from carob bean gum
  • Ext+GLMN+FOS is the extract (average results of Batch I and Batch II) together with galactomannans from carob bean gum and fructooligosaccharides.
  • Table 2b Analysis of the one-way variance by comparing the F test statistic of the bioaccesibility of reducing sugar and bioactive components after in vitro digestion of carob extracts, together with galactomannans and FOS.
  • the extracts and their corresponding mixtures with galactomannans and FOS were initially analyzed to quantify their content in TPP by means of the Folin Ciocalteu method and also after digestion.
  • TPP content of the Batch I and Batch II carob extracts is 4.21 and 4.29% respectively (4.3 % as average).
  • TPP were loss almost completely, reaching values between 86 (4.21-0.59%) and 98% (4.29-0.10%) respectively (from 4.3 to 0.4 % as average, 91 % of TPP loss)
  • the extracts and their corresponding mixtures with galactomannans and FOS were assessed for their antioxidant capacity before and after digestion. As shown in Table 2a, the antioxidant capacity followed similar trend to the Total Polar Phenols content as expected due to their direct correlation.
  • the antioxidant capacity expressed by Trolox ratio was 12.8%, and after digestion, was reduced by approximately 88% (12.8-1 .6%), and for the Batch II extract, a 71 % (12.8-3.7%); as average, the antioxidant capacity was reduced in the extract about 80 % (from 12.8 to 2.6 %).
  • the extract was added the locust bean gum with GLMN, the extract followed the same trend, reducing its antioxidant capacity as average about 50 % (from 7.2 to 3.8 %).
  • the antioxidant capacity showed an average reduction of about 70 % (from 5.5 to 1 .6 %).
  • the addition of GLMN and FOS to the final composition reduced the effect of the digestion process on the antioxidant capacity between 30 and 10 % respectively compared to the initial extract.
  • Example 3 Physiological effects of the composition.
  • mice Three groups of C57BL/6J 16-week old male mice were used:
  • mice 12: Fed ad libitum with a standard chow
  • Said composition was obtained according to the method of Example 1 and comprises 36% wt. of GLMN, 3% wt. of FOS and 1 % wt. of the phenolic extract from carob.
  • mice fed the high fat/high sucrose diet gained more weight during the 12-week period compared to controls (P ⁇ 0.001 ), regardless of the treatment with the composition of the invention.
  • the consumption of said composition together with a high fat/high sucrose diet for 12 weeks is not able to reduce body weight gain.
  • HFD mice showed increased plasma levels of triglycerides (PO.01 ; Figure 3A), total lipids (PO.01 ; Figure 3B), total cholesterol (PO.001 ; Figure 3C), c-LDL (PO.01 ; Figure 3D) and c-HDL (PO.05; Figure 3E), compared to controls.
  • Treatment with the composition of the invention for 12 weeks did not have an impact neither in the plasma triglycerides, nor in total lipids. However, it significantly reduced the concentrations of total cholesterol (P ⁇ 0.05) and c-LDL (p ⁇ 0.05) in plasma and increased c-HDL (P ⁇ 0.05).

Abstract

The invention relates to a composition comprising galactomannan in an amount ranged from 36 to 40% w/w, fructooligossacharides in an amount of 2 to 4% (w/w) and at least one extract of polar nature containing bioactive compounds, with a concentration between 30 and 49% in the composition. The invention also relates to the uses of said composition for the administration of said bioactive compounds contained in the extract, protecting from their degradation during the physiological digestion.

Description

DIETARY FIBER COMPOSITION FOR PROTECTING BIOACTIVE COMPOUNDS DURING THE PHYSIOLOGICAL DIGESTION PROCESS.
TECHNICAL FIELD OF THE INVENTION
The invention relates to a composition comprising galactomannans, fructooligosaccharides (FOS) and bioactive compounds of polar nature, mainly, phenols and polyphenols from carob {Ceratonia siliqua L) pod extract. The adequate ratio of galactomannans (soluble fibers) and fructooligosaccharides (insoluble fibers) protects the bioactive compounds of polar nature against degradation during digestion, increasing their bioavailability thereof in the organism. Thus, the present invention refers to the field of administration of bioactive compounds.
BACKGROUND ART
There are many documents regarding food products that contain several kinds of fibers and additional ingredients, such bioactive compounds, which can be used to prevent or to treat different pathologies or to promote a well-being state. See for example:
International patent application WO2013/132456 relates to prebiotic mixtures comprising at least one plant polysaccharide fibre and at least one vegetal extract, for use in the prevention and/or the treatment of gastrointestinal pathologies that exhibit an alteration of the intestinal flora balance; wherein said fibre is between 50 and 90% and said extract is between 5 and 25% by weight of said mixture;
International patent application WO2012/044242 relates to a food product containing viscous fibres (including carob gum) between 4 and 35% by dry weight. Additionally, one or more components selected from the group of indigestible carbohydrates (for example fructooligosaccharides), polyphenols, fatty acids and probiotics can be added. The claimed food product may improve glucose tolerance/insulin sensitivity already in the late post-prandial phase following acute meal.
International patent application WO201 1/060123 regards nutritional compositions comprising fructooligosaccharides in an amount of 35 to 44% by weight; a polysaccharide that is not a partially hydrolyzed guar gum such as, for example, an arabinogalactan in an amount of 50 to 38% by weight; and inulin in an amount of 12 to 24% by weight. The fructooligosaccharides and the polysaccharide may be present in a weight ratio of about 1 :1. More specifically, the fructooligosaccharides and inulin may be present in a weight ratio of about 7:3. These compositions promote gut microbiota balance and health. International patent application WO2006/134409 refers to synergistic compositions of prebiotic components selected from fructose polymers, either containing a glucose end-group, or without a glucose end-group, and one or more components of modified or unmodified starches and partially hydrolysed inulin, natural oligofructoses, fructooligosaccharides, lactulose, galactomannans, indigestible polydextrose, acemannan, vanous gums, indigestible dextrin and partial hydrolysates thereof, transgalactooligosaccharides (GOS), xylooligosaccharides (XOS), beta-glucans and partial hydrolysates thereof, together if desired with phytosterol/phytostanol components and their suitable esters, and if desired other plant extracts, mineral components, vitamins and additives.
Rutz et al. (Carbohydrate Polymers 98 (2013) 1256-1265) disclose a solution to encapsulate active substances like carotenoids and phenolic compounds from purple Brazilian cherry juice, using a combination of the polysaccharides xantham gum and galactomannan.
Patent application US2003/0059458 discloses a composition of water insoluble carob fibre in an amount of about 10 to about 90% by weight and an encapsulating material of at least one water soluble dietary fiber in an amount of about 10 to about 90%, being the water soluble dietary fibre a natural or a chemically modified polysaccharide.
However, in most of the cases, most of the bioactive compounds are lost during digestion, decreasing the final available amount for the consumer. Furthermore, the compositions that specifically intend to protect bioactive compounds during digestion, mainly by using polysaccharide carriers, contribute to the increase of blood glucose content of diabetic subjects. Accordingly, there is a need for providing compositions not only rich in bioactive compounds of polar nature but also that have increased bioavailability thereof and, at the same time, that reduce blood glucose content after consumption. DETAILED DESCRIPTION OF THE INVENTION
The inventors of the present invention have discovered that when a composition comprises galactomannan (soluble fibers), hereinafter GLMN, in an amount of 36 to 40% by weight, based on the total weight of the composition and fructoligosaccharides, hereinafter FOS, (non-soluble fibers) in an amount of 2 to 4% by weight, based on the total weight of the composition, the extract of polar nature present in said composition are, surprisingly, protected from being digested in the stomach (see example 2), avoiding the almost complete loss of the bioactive compounds contained in the extract during the digestion process, and resulting in a higher bioavailability of these compounds after digestion. As a consequence, the amount of bioactive compounds to be administered can be decreased because there is a low loss thereof during digestion. Furthermore, the inventors have also discovered that, not only the bioactive compounds included in the extract of polar nature are present after digestion, but also soluble and insoluble fibre, and therefore, the composition of the present invention may play a role to prevent from risk factors that commonly cluster together (like dyslipidaemia, hypertension and hyperglycaemia) and have been termed as metabolic syndrome.
Recently, the National Cholesterol Education Program's Adult Treatment Panel III report (ATP III) defined criteria used to identify patients with the metabolic syndrome. ATP III identified some risk factors of the metabolic syndrome that relate to cardiovascular disease (CVD): abdominal obesity, atherogenic dyslipidaemia, raised blood pressure, insulin resistance, glucose intolerance, proinflammatory and prothrombotic states. The pathogenesis of the metabolic syndrome is not known, but there seem to be three potential etiological categories: obesity and disorders of adipose tissue, insulin resistance and a number of independent factors that mediate specific components of the metabolic syndrome (Beilby, J. 2004, Clin Biochem Rev. 25(3): 195-198). Therefore, due to the protective effect associated to the combination of GLMN and FOS over the extract of polar nature, these compounds are useful for the manufacture of a composition of soluble and insoluble fibres for the administration of several functional molecules and/or for the development of nutraceuticals more effective at even lower doses. In the present invention, an adequate composition of soluble and insoluble fibres is presented as a natural "encapsulating carrier" to protect bioactive compounds of polar nature, such as phenolic compounds, from digestion process and therefore to improve their bioavailability and beneficial effect on the organism.
Thus, in a first aspect the invention relates to a composition, hereinafter "composition of the invention", comprising GLMN in an amount of from 36 to 40% by weight, based on the total weight of the composition, a FOS in an amount of from 2 to 4% by weight, based on the total weight of the composition, and at least one extract of polar nature.
In the context of the present invention, the term "composition" refers to any substance comprising any combination of three or more molecules, wherein said molecules are, at least, GLMN, a FOS and an extract of polar nature containing bioactive compounds such as phenolic compounds.
The term "galactomannan" refers to a polysaccharide consisting of a mannose backbone with galactose side groups, more specifically, a (1 -4)-linked β-D- mannopyranose backbone with branchpoints from their 6-positions linked to a-D- galactose, i.e. 1 -6-linked a-D-galactopyranose, as represented in Figure 1. Galactomannan can be isolated from multiple sources. There are four major sources of seed galactomannans: locust bean (Ceratonia siliqua), guar (Cyamopsis tetragonoloba), tara (Caesalpinia spinosa Kuntze), and fenugreek (Trigonella foenum- graecum L). Depending on the mannose:galactose ratio of the polymer, examples of galactomannans include, without limiting to, fenugreek gum (mannose:galactose -1 :1 ), guar gum (mannose:galactose ~2:1 ), tara gum (mannose:galactose -3:1 ) and locus bean gum or carob gum (mannose:galactose -3:1 ). Any galactomannan can be used in the composition of the invention. In a particular embodiment, the galactomannan comprises a mannose:galactose ratio of about 3:1 from carob (Ceratonia siliqua L) gum.
The source of galactomannan is the endosperm of carob seed (Ceratonia siliqua L) ground with an amount of galactomannan no less than 75% (dry weight) (see Figure 2). In a particular embodiment, the amount of galactomannan in the final fiber complex (composition) of the present invention is 36% by weight based on the total weight of the composition. The fructooligosaccharide refers to oligofructose produced by partial enzymatic hydrolysis of chicory inulin. Oligofructose consist of fructose units linked by β(2-1 ) linkages. The degree of polymerization chiefly ranges between 2 and 8. In another particular embodiment, the FOS comprises no less than 90% by weight of oligofructose (dry matter). In a particular embodiment of the composition of the invention, the amount of fructooligosacharide is 3% by weight based on the total weight of the composition.
Thus, in a particular embodiment, the amount of GLMN is 36% by weight based on the total weight of the composition and/or the amount of FOS is 3% by weight based on the total weight of the composition.
As explained above, the present invention is based on the fact that the presence of GLMN and FOS in a particular amount protects the bioactive compounds contained in the extract of polar nature present in a composition from digestion process.
In the present inventions, the term "extract" refers to a substance obtained from plants by extracting a part of a raw material, often by using a solvent such as ethanol, water or a combination of both.
In the present invention, the term "bioactive compounds" refers to the ingredients or components which are biologically active, i.e. are responsible for conferring to the composition therapeutic properties, and these compounds are soluble in water or in mixtures of water and ethanol [not more than 20% of ethanol (v/v) in the final solvent mixture]
In a particular embodiment of the composition of the invention, the presence of fibres in the final product protects the bioactive compounds contained in the extract of polar nature during the digestion process, chiefly phenolic compounds. Any natural extract of polar nature can be used in the context of the present invention. Various extracts and plant powders are incorporated into the composition of the invention depending on the desired properties according to the end use of said compositions.
Examples of extracts with therapeutic properties which can be used in the composition of the invention include, without being limited to, botanical extracts standardized to bioactive compounds, such as, one or more of those of Panax ginseng (red, Korean ginseng), Panax ginseng (white, Chinese ginseng), Rhodiola rosea (golden root), Panax quinquefolium (American ginseng), Eleutherococcus senticosus (Siberian ginseng), Cynara scolymus (artichoke), Uncaria tomentosa (Cat's claw), Lepidium meyenii (maca, Peruvian ginseng), Paullinia cupana (guarana), Croton lechleri (Sangre de Grado), Whitania somnifera (ashwagandha, Indian ginseng), Panax japonicus (Japanese ginseng), Panax vietnamensis (Vietnamese ginseng), Panax trifolius, Panax pseudoginseng, Panax notoginseng, Malpighia glabra (acerola), Ylex paraguayiensis (Yerba mate), Astragalus membranaceus (astragalus), Stevia rebaudiana (stevia), Pfaffia paniculata (Brazilian ginseng, suma), Ginkgo biloba, Tabebuia impetiginosa (Pau d'arco), Echinacea purpurea, Peumus boldus (boldo), Gynostemma pentaphyllum (Jiaogulan, also known as Southern Ginseng or Xiancao), Sutherlandia frutescens (African ginseng), Aloe vera (aloe), Cistanche salsa, Cistanche deserticola (and other Cistanche sp.), Codonopsis pilosula ("poor man's ginseng."), Nopal opuntia (Prickly pear cactus), Citrus sinensis (Citrus aurantium) and other members of the citrus family (lemon, lime, tangerine, grapefruit), Camelia sinensis (tea), Plantago psyllium (psyllium), Amaranth edulis and other amaranth sp. (amaranth), Commiphora mukul (guggul lipid), Serenoa repens, Serenoa serrulata (saw palmetto), Cordyceps sinensis (Cordycaps), Lentinula edodes (Shitake), Ganoderma lucidium (Reishi), Grifola frondosa (maitake), Tremella fuciformis (Silver ear), Poria cocos (Hoelen), Hericium erinaceus (Lion's Mane), Agaricus blazei (Sun mushroom), Phellinus linteus (Mulberry yellow polypore), Trametes versicolor, Coriolus versicolor (Turkey tails), Schizophyllum commune (Split gill), Inonotus obliquus (Cinder conic), oat bran, rice bran, linseed, garlic, Ceratonia siliqua (locust been gum or flour from the seeds of carob tree), Cyanopsis tetragonoloba (guar gum, EU Food additive code E412), Xanthomonas campestris (xanthan gum), Olea europaea (olive) and related herbal extracts with bioactive compounds of polar nature
In a particular embodiment, the extract of polar nature is derived from carob, particularly, from carob pods.
The extracts used as bioactive compounds in the composition of the invention can include multiple antioxidant compounds such as phenols, polyphenols, secoiridoids, peptides, carotenoids, enzymes, gluthatione, hormones, lipids, minerals, vitamins, saponines, steroids, etc. Examples of phenols include, but without being limited to, simple phenolic acids and derivatives, hydroxycinnamic acids and derivatives, flavonoids (flavones, isoflavones, flavanones, flavonols, anthocyanins), tannins (gallotannins and ellagitannins), and related components of polar nature. In a particular embodiment, the extract of polar nature included in the composition of the invention comprises hydrolysable tannins derived from gallic acid. Preferably, these compounds may represent between 50 to 90% by weight of the extract of polar nature, and more preferably 70% by weight. The amount of the extract of polar nature in the composition of the invention can encompass a wide range of concentrations. Nevertheless, in a particular embodiment, the composition comprises an amount of the extract of polar nature from 30 to 49 % by weight based on the total weight of the composition, preferably 40% by weight. The composition of the invention may be a pharmaceutical or a nutritional composition. The pharmaceutical and the nutritional composition may be formulated, but not limited to, in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, microcapsule, powder, granule, ointment, solution, paste, suppository, injection, inhalant, gel, microsphere or aerosol. In a particular embodiment, the composition is formulated for oral administration.
The composition of the invention comprises a prophylactically or therapeutically effective amount of at least one bioactive compound of polar nature. The term "prophylactically effective amount", as used herein, means that the bioactive compound contained in the composition is in sufficient quantity to exert its effect on the subject. The effect will depend on the kind of the bioactive compound used in the composition of the invention. Examples of bioactive compounds have been disclosed above.
Furthermore, compositions of the present invention may include a carrier. Depending on the kind of compositions of the present invention, a carrier may be a dietary suitable carrier or a pharmaceutically acceptable carrier, as long as it is compatible with the bioactive principle of the composition. Examples of a dietary suitable carrier include, but are not limited to, dietary suitable excipients and diluents. The term "excipient" relates to a substance that aids the absorption of any of the bioactive compound of the composition of the invention, stabilizes said bioactive compound or aids the preparation of the composition in the sense of giving consistency or contributing flavours that make it more enjoyable. Thus, excipients may have the function of holding the components together, such as starches, sugars or celluloses, a sweetening function, colouring function, drug protection function, the function of filling a tablet capsule, sachets, softgels and related galenic forms. Therefore, the term "excipient" is defined as the material included in the galenic forms, is added to the bioactive compounds or their associations to enable their preparation and stability, modify their organoleptic properties or determine the physical-chemical properties of the pharmaceutical composition and its bioavailability.
The term "nutritional composition" relates to a food or a dietary supplement that regardless of providing nutrients to the subject has a beneficial effect on one or more health functions, so as to provide a better state of health and well-being. Accordingly, such nutritional composition may be destined for the prevention and/or treatment of a disease or for the reduction of disease risk factors, such as those related wit metabolic syndrome.
The inventors of the present invention have discovered that the presence of GLMN and FOS in a particular amount protects the bioactive compounds of polar nature present in a composition from being digested. In this way, the bioactive compounds can be administered in a lower dose for exerting its therapeutic effect in a subject which, combined with the soluble and insoluble fibres of the composition, can be used for treating and/or preventing any disease in a more effective way. Consequently, in another aspect, the present invention relates to the composition of the invention for use as a medicament. Depending on the therapeutic properties of the bioactive components, e.g. the extract, the present composition can be used for treating and/or preventing a particular disease. In a particular embodiment, when the bioactive compounds are coming from a carob extract, particularly, carob pod extract, the invention relates to a composition for use in the treatment and/or prevention of risk factors related to metabolic syndrome.
The term "treatment", as understood herein, relates to fight the effects of a disease or pathological condition of interest in a subject (preferably a mammal and, more preferably, a human) that includes: (i) inhibiting the disease or pathological condition, i.e. arresting its development;
(ii) relieving the disease or the pathological condition, i.e. causing regression of the disease or the pathological condition or its symptoms;
(iii) stabilizing the disease or pathological condition.
The term "prevention" as understood in the present invention consists of preventing the onset of the disease, that is, preventing the development of disease or pathological condition from occurring in a subject (preferably a mammal and, more preferably, a human), particularly when said subject is predisposed to develop the pathological condition.
The term "metabolic syndrome" refers to the set of metabolic alterations that jointly increase the risk of diabetes and cardiovascular disease, including the combination of obesity, dyslipidaemia (for example, triglycerides and hypercholesterolemia) and hyperglycaemia.
In view of the properties shown by the combination of a galactomannan and FOS as explained above, the present invention relates to the composition for use in increasing the absorption of an extract of polar nature in a subject with the following composition: - galactomannans in an amount of from 36 to 40% by weight based on the total weight of the composition,
fructooligossacharides in an amount of from 2 to 4% by weight based on the total weight of the composition, and
said extract of polar nature.
The terms galactomannan, FOS and extract of polar nature have been explained in previous paragraphs.
In a particular embodiment, the amount of galactomannan is 36% by weight based on the total weight of the composition and/or the amount of fructooligosascharide is 3% by weight based on the total weight of the composition.
In another particular embodiment, the galactomannan comprises a mannose:galactose ratio of about 3:1. In another particular embodiment, the fructooligossacharides comprise no less than 90% by weight of oligofructose (%, dry matter).
In another particular embodiment, the extract of polar nature comprises hydrolysable tannins derived from gallic acid. In a more particular embodiment, the hydrolysable tannins derived from gallic acid represent between 50 to 90% by weight of the extract of polar nature, preferably 70% by weight.
In another particular embodiment, the extract of polar nature is derived from carob, preferably from carob pods.
In another particular embodiment, the extract of polar nature is in an amount of from 30 to 49% by weight based on the total weight of the composition, preferably 40% by weight.
In another particular embodiment, the extract of polar nature is a mixture of one or more botanical extract different from carob extract. Examples of botanical extracts have been disclosed previously in the present disclosure. In another aspect, the invention relates to a method for obtaining the composition of the invention, hereinafter "method of the invention", comprising mixing an extract of polar nature with (i) galactomannan in an amount of from 36 to 40% by weight based on the total weight of the composition and (ii) FOS in an amount of from 2 to 4% by weight based on the total weight of the composition.
In a particular embodiment of the method of the invention, the amount of galactomannan is 36% by weight based on the total weight of the composition and/or the amount of FOS is 3% by weight based on the total weight of the composition. In another particular embodiment of the method of the invention, the galactomannan comprises a mannose:galactose ratio of about 3:1.
In another particular embodiment of the method of the invention, the galactomannan comes from carob seeds and/or the FOS comes from inulin. In another particular embodiment of the method of the invention, the extract of polar nature is carob extract, particularly, carob seed extract or carob pod extract.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or stages. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention. DESCRIPTION OF THE DRAWINGS
Figure 1 discloses a segment of galactomannans showing mannose backbone (below) with a branching galactose unit (top), wherein m>1 and n>1. Figure 2 shows the chemical structure of carob galactomannans. It is a linear chain of 1 ,4-β-ϋ mannopyranose units with 1 ,6-linked a-D galactopyranose units attached to every fifth mannose unit.
Figure 3 shows a manufacturing flow chart of the carob (Ceratonia siliqua L) pod extract.
Figure 4 shows the body weight gain of mice fed a standard chow (Controls; n=12), a high fat/high sucrose diet (HFD; n=12) or a high fat/high sucrose diet supplemented with invented composition (HFD+Composition; n=12) for twelve weeks. Values are represented as the meaniSEM. *** P<0.001 vs Controls.
Figure 5 shows Glycemia (A), Insulin plasma concentrations (B) and HOMA-IR (C) in mice fed a standard chow (Controls; n=12), a high fat/high sucrose diet (HFD; n=12) or a high fat/high sucrose diet supplemented with the composition of the present invention (HFD+Composition; n=12) for twelve weeks. Values are represented as the mean+SEM. ** P<0.01 vs Controls; * P<0.05 vs Controls; # P<0.05 vs HFD.
Figure 6 shows the lipid profile of mice fed a standard chow (Controls; n=12), a high fat/high sucrose diet (HFD; n=12) or a high fat/high sucrose diet supplemented with the Composition+ (HFD+Composition; n=12) for twelve weeks. Values are represented as the mean+SEM. *** PO.001 vs Controls; ** P<0.01 vs Controls; * PO.05 vs Controls; # P<0.05 vs HFD. Figure 7 shows reactive hyperemia measured by laser speckle from the feet of mice fed a standard chow (Controls; n=12), a high fat/high sucrose diet (HFD; n=12) or a high fat/high sucrose diet supplemented with the Composition+ (HFD+Composition; n=12) for twelve weeks. Black line marks the basal blood flow. Values are normalized to 100% with their basal blood flow and represented as the meaniSEM.
Figure 8 shows the Hyperemia/lschemia ratio calculated by the Area Under the Curve of the reactive hyperemia of mice fed a standard chow (Controls; n=12), a high fat/high sucrose diet (HFD; n=12) or a high fat/high sucrose diet supplemented with the Composition+ (HFD+Composition; n=12) for twelve weeks. Values are represented as the meaniSEM. *** PO.001 vs Controls; # P<0.05 vs HFD.
EXAMPLES
Example 1. Obtaining of the composition of the invention
I. MATERIAL AND METHODS Samples
The samples tested comprised carob extract as an extract of polar nature, galactomannans from locust bean gum (>75% galactomannans) and fructooligosaccharides (>90% purity).
Plant material
Deseeded and chopped carob pods were obtained, ground and then extracted according to the flow chart (Figure 3), mixed and packaged in the processing factory of Pharmactive (Madrid, Spain). The carob kibbles were stored in a cool and dry place until analysis and extraction process.
Polar phenols extraction from carob pods
A general scheme illustrating the extraction process can be observed in Figure 3. The ground raw material was extracted by a mixture of water and ethanol, where the presence of ethanol was not more than 20% in the final solvents mixture.
Temperature control is a critical parameter for the extraction process. It should be limited up to 85°C in order to avoid the deterioration of some phenolic compounds. Organic solvents such as ethyl acetate, hexane, petroleum ether, acetone, methanol or the like are not suitable for carob pod extraction.
The crude extract is a dense liquid, with intense brown colour and characteristic aroma of carob (Ceratonia siliqua L). Once cooled to room temperature the extract is concentrated and dried, using different drying techniques.
The dry solid extract is then milled; the last step comprised of adjusting the particle size by milling and grinding the dry extract in a Hammer or blades mill with a strict temperature control to avoid bioactive compound degradation. Finally, the powder solid extract is sieved through a light sieve to obtain a maximum particle size of 240 μηη.
The obtained extract was characterized by HPLC, showing the following profile of components:
Table 1 : Identified individual polar phenolic compounds in carob pod extract and their retention time (tR), UV-VIS spectra, quasi-molecular [M-H]" and fragment ions, acquired in negative ionisation mode. tR Molecule UV-VIS [M-H]- Fragment ions
(min) (nm) (m/z) (m/z)
4.2 Gallic acid 229/274 169.0 125.1
Monogalloyl 227/278 331.0
4.5 169.1
hexoside
Monogalloyl 226/278 331.0
4.8 168.9
hexoside Molecule UV-VIS [M-H]- Fragment ions
226/276 635.0 483.0, 331.0, 313.0, 271.0
Trigalloyl hexoside
211.0, 169.1
Digalloyl hexoside 227/275 483.0 331.0, 313.0, 169.0
226/275 483.0 443.0, 331.0, 313.0, 210.9,
Digalloyl hexoside
169.0, 124.1
Digalloyl hexoside 226/277 483.0 331.0, 313.0, 169.0
Digalloyl hexoside 228/280 483.0 331.1
224/273 635.0 483.0, 464.7, 331.0, 312.9,
Trigalloyl hexoside
271.0, 210.9, 168.9
267 635.0 483.0, 331.0, 312.9, 270.8,
Trigalloyl hexoside +
443.0 210.9, 168.9
unknown
280 634.8 482.9, 331.0, 313.0, 271.0,
Trigalloyl hexoside
241.1, 211.1, 168.9
224/272 635.0 483.0, 464.9, 331.0, 313.0,
Trigalloyl hexoside
271.0, 241.1, 211.1, 168.9
224/270 635.8 483.0, 464.9, 422.9, 331.0,
Trigalloyl hexoside 313.0, 270.9, 241.0, 210.9,
193.1, 168.9
224/272 635.1 483.0, 464.9, 422.9, 331.0,
Trigalloyl glucoside 313.0, 271.0, 240.8, 210.9,
193.1, 168.9
224/278 634.9 483.1, 465.0, 421.0, 331.0,
Trigalloyl hexoside
313.0, 271.0
Tetragalloyl 223/272 786.9 635.0, 483.0, 330.8, 313.0, hexoside 271.0, 211.0, 168.9
223/272 634.5 483.1, 465.0, 443.0, 313.0,
Trigalloyl hexoside
168.9
225/275 787.1 635.0, 483.1, 465.8, 443.0,
Tetragalloyl
423.0, 313.0, 271.1, 168.9, hexoside
151.1
224/278 786.9 635.0, 483.0, 465.8, 449.0,
Tetragalloyl
313.0, 271.0, 210.7, 168.9, hexoside
121.1
226/285 635.0 483.1, 465.8, 449.0, 401.1,
Trigalloyl hexoside
313.0, 271.0, 210.7
Trigalloyl hexoside 225/276 634.9 482.8,465.0,313.0
Tetragalloyl 223/271 787.0
635.0, 482.9, 422.9 hexoside
Tetragalloyl 223/274 787.0
635.0,482.9,464.9, 168.9 hexoside
Tetragalloyl 223/270 786.8
635.0,465.8,403.0, 124.1 hexoside tR Molecule UV-VIS [M-H]- Fragment ions
Tetragalloyl 263/278/370 786.9
37.2 635.0, 464.9
hexoside + 479.0
317.0
Myricetin hexoside
Tetragalloyl 223/278 786.9 635.0, 401 .0, 313.0, 210.9,
40.0
hexoside 169.0
Tetragalloyl 225/276 787.0
45.1 635.0, 617.0, 465.8
hexoside
Tetragalloyl 223/281 /393sh 786.9 635.0, 616.9, 462.8, 271 .0,
47.3
hexoside 124.8
Tetragalloyl 223/277/355 787.0
hexoside + 463.0 617.0, 464.9, 271.0, 168.8
49.5
Myricetin deoxy- 316.0
hexoside (1 )
Myricetin deoxy- 260/300sh/348 463.0
51 .3 316.9, 315.9, 287.0, 271 .0 hexoside (2)
Ellagic acid 250/369 463.0
52.1 301.0, 229.0, 201 .1 , 169.1 hexoside
54.2 Quercetin hexoside 253/298sh/352 462.9, 300.9, 270.8, 254.9, 151 .1
56.5 Naringin 225/281/335 579.0 271 .0
Isohesperetin 229/281/328 579.0
58.6 459.1 , 271 .0
(Nari rutin)
Quercetin deoxy- 226/253/261 sh/ 447.0
59.8 300.0, 301 .0, 271 .0, 255.0 hexoside 348
Hesperetin 226/283/331 609.1
62.2 461 .1 , 446.9, 300.9
rutinoside
Kaempferol 221/251/263sh/ 593.1
71 .0 285.0
rutinoside 345
220/253/294sh/ 314.9
72.7 Methylquercetin 299.9, 271 .0
354
In this way, two different batches were obtained from carob pod extracts, called Batch I and Batch II, enriched in bioactive compounds of polar nature. Then Batch I or Batch II are mixed with the soluble/insoluble fibres complex (galactomannans from carob seeds and FOS from inulin) in order to obtain the final product (composition) of the present invention. The final amounts of the carob pods extract, galactomannans and FOS in the compositions are, respectively, 40, 36 and 3%, by weight, based on the total weight of the composition. Due to the homogeneity of the composition of the present invention, the results obtained with Batch I and Batch II were considered together as can be observed in Table 1 and also in Table 2a for the statistical analyses during the digestion process. The final product (compositions obtained from two different batches of carob extracts denominated Batch I and Batch II together with GLMN and FOS) was analysed before and after in vitro digestion for bioaccesibility of bioactive compounds: total polar phenols with antioxidant capacity, together with glycaemic sugars (Table 2a).
Example 2. In vitro digestion protocol
To demonstrate the protective effect of the composition of the invention during the physiological digestion process, an in vitro digestion protocol was established. This protocol comprises the following three phases: 1 ) cephalic or salivary phase, 2) gastric phase, and 3) intestinal or duodenal phase. All processes were carried out in one and the same tube.
Preparation of in vitro digests
An in vitro abiotic digestion was carried out, comprising three stages (oral, gastric and intestinal) as described by Hollebeeck et al., 2013. Briefly, the salivary step was performed with 3.9 units of α-amilase/mL of sample, at pH 6.9, 37°C, 5 min of incubation, and aerobic conditions. The gastric step was performed with 71 .2 units of pepsin/mL of sample at pH 2, 37°C, 90 min of incubation, and anaerobic conditions while the abiotic duodenal step was run with a mixture of 9.2 mg of pancreatin and 55.2 mg of bile extract/mL of sample at pH 7, 37°C and 150 min, under anaerobic conditions. The final mixture was centrifuged at 5000 rpm for 40 min, at 4°C. A soluble fraction was obtained by centrifugation which was freeze-dried. The resulting powder was stored at -20°C until analysis. This process is performed in duplicate, and the corresponding digested samples are analysed in triplicate.
Total polar phenolic compounds
Folin-Ciocalteu adapted to a micromethod format was the test selected for analysis of total polar phenolic compounds in the different samples, before and after the digestion process (Contine, Baccelini, Massantini & Anelli, 2008). The reaction was initiated by mixing 10 μΙ_ of sample with 150 μΙ_ of Folin-Ciocalteu reagent. After incubation at room temperature during 3 min, 50 μΙ_ of sodium bicarbonate solution were added. The kinetics of the reaction at 37 °C was followed for 120 min by measuring the absorbance at 735 nm once every minute. A gallic acid calibration curve was used for quantification. Results are expressed as percentage (% by dry weight). All measurements were performed in triplicate.
Total antioxidant capacity
ABTS' decolourisation assay was performed according to Oki, Nagai, Yoshinaga and Nishiba (2006). An ABTS'+ aqueous solution was prepared by adding 140 mM potassium persulfate (44 μΙ_) to a 7 mM ABTS'+ aqueous solution (2.5 ml_), and the mixture was then allowed to stand for 16 h at room temperature. The working solution of the radical ABTS'+ was prepared by diluting the stock solution 1 :75 (v/v) in 5 mM sodium phosphate buffer pH 7.4 to obtain an absorbance value of 0.7±0.02 at 734 nm. Samples (30 μΙ_) were added to 270 μΙ_ working solution ABTS'+ in a microplate. The absorbance was measured at 734 nm for 10 min at 30°C with measurements every 2 min. After 5 min, the reaction was complete. Trolox and Gallic acid calibration curves were used for quantification. Results were expressed as % (dry weight). All measurements were performed in triplicate.
Insoluble and soluble fibres
Insoluble and soluble (galactomannan) fibres were determined using respectively the "Total Dietary Fibre Assay Kit" and the "Galactomannan Kit" (Megazyme International Ireland, Ireland), based on an enzymatic-gravimetric method described in the manufacturer's instructions. Results were expressed as % (dry weight).
Moisture
Prior to analysis, the capsules with the composition of the invention employed for digestion were assessed for their moisture content. For this, samples of about 1 g of the solid powder were heated at 1 10°C for 15 min and cooled in a desiccator to room temperature. The moisture content was determined by the gravimetric method according to the AOAC-925.10. The samples were weighted accurately (~1 g), and dried until constant weight in an oven at 105°C. Results were expressed as %.
Glycaemic sugars
Glucose, mannose and fructose were determined employing the Megazyme kit according to the manufacturer's instructions (Megazyme KMANGL, Ireland) adapted to micro-method format. A Bio Tek Power Wave TM XS microplate reader (Bio Tek Instruments, USA) was used to measure the samples absorbance at 340 nm. D- mannose, D-glucose, D-mannose and D-fructose contents in each sample were expressed as % of glucose, mannose and fructose (dry weight). These measures showed that the compositions of the invention contain reducing sugars but, surprisingly, are capable of regulating the levels of blood sugar as it will be demonstrated in the in vivo assays of Example 3.
Statistical analysis
Reducing sugars together with the polyphenolic content and antioxidant capacity data were analysed using the SPSS 17.0 statistical software (SPSS Inc., Chicago, IL).
II. RESULTS
Bioaccessibility of bioactive compounds A tracing of the glycaemic sugars presented in GLMN and FOS as well as the polyphenols present in the bioactive extract of the composition was carried out during the digestion of the samples in order to demonstrate the protective effect of the composition obtained. 1. Glycaemic sugars
As shown in Table 2a, initially the glucose content of both samples was similar, between 5.6 and 4.8% for the Batch I and Batch II samples respectively (5.2% as average), and that after digestion about 2-fold increase was obtained (respectively 12.1 and 9.8%, 1 1 .0 % as average). The same feature was also observed for the case of fructose, which increased after digestion about 2-folds for both batches (as average from 6.4 to 12.2 %).
When carob gum with galactomannan (GLMN) is included in the formula, the release of simple sugars is also favoured after digestion. In the case of glucose, the increase is about 3.5 folds (from 3.1 to 10.8 % as average) A similar result was obtained for fructose, which concentration increased about 3-fold after digestion process (from 3.8 to 1 1.5 as average).
The addition of 3% of FOS to the above formula showed similar results regarding reducing sugars concentration after digestion. In fact, the release of glucose after digestion as about 3-fold higher concertation compared to the initial composition (from 2.6 to 8.2 % as average), and same trend was observed for fructose which increased its concentration about 2 folds after digestion (from 4.6 to 9.7 % as average). Table 2a. Initial composition and corresponding bioaccesibility of reducing sugars and bioactive compounds after in vitro digestion of carob extracts, together with galactomannans and FOS.
Initial samples
Extract Ext+GLMN Ext+GLMN+FOS
Glucose 5.2±0.6 3.1 ±0.2 2.6±0.4
Fructose 6.4±0.9 3.8±0.2 4.6±0.1
TPP 4.3±0.1 2.0±0.1 2.2±0.1
Antioxidant
12.8±0.1 7.2±0.0 5.5±0.9
Capacity
Digested samples
Parameters
Extract Ext+GLMN Ext+GLMN+FOS
Glucose 1 1.0±1.6 10.8±0.7* 8.2±0.4
Fructose 12.2±3.3 1 1.5±0.4* 9.7±0.0
TPP 0.4±0.2 0.2±0.0* 1 .1 ±0.1
Antioxidant
2.6±1.4 3.8±1 .4 1 .6±0.2
Capacity Ext+GLMN is the extract (average results of Batch I and Batch II) together with galactomannans from carob bean gum
Ext+GLMN+FOS is the extract (average results of Batch I and Batch II) together with galactomannans from carob bean gum and fructooligosaccharides. Table 2b. Analysis of the one-way variance by comparing the F test statistic of the bioaccesibility of reducing sugar and bioactive components after in vitro digestion of carob extracts, together with galactomannans and FOS.
"F" values
Extract (F; p) Ext+GLMN (F; p) Ext+GLMN+FOS (F; p) Glucose 22.3; 0.04 220.4; 0.00 250.9; 0.00
Fructose 20.9; 0.04 533.9; 0.00 10609.0; 0.00
TPP 247.4; 0.00 1074.1 ; 0.00 199.9; 0.00
Antioxidant
94.1 ; 0.01 1 1.4; 0.08 33.6; 0.03
Capacity
Mean values with higher Anova F-ratio and significance p <0.05 (95%) indicate statistical differences between the initial composition and after digestion process. 2. Total polar phenols (TPP) content
The extracts and their corresponding mixtures with galactomannans and FOS were initially analyzed to quantify their content in TPP by means of the Folin Ciocalteu method and also after digestion. As shown in Table 2a, initially the TPP content of the Batch I and Batch II carob extracts is 4.21 and 4.29% respectively (4.3 % as average). After undergoing in vitro digestion, TPP were loss almost completely, reaching values between 86 (4.21-0.59%) and 98% (4.29-0.10%) respectively (from 4.3 to 0.4 % as average, 91 % of TPP loss)
The addition of galactomannans to the extract followed similar trend, the decrease in TPP content was very important after digestion, approximately 90% for Batch I + galactomannan (2.06-0.2%) and also Batch II + galactomannan (1 .95-0.19%; on average about 90 % (from 2.0 to 0.2 %, Table 2a)
However, when FOS are added to the blend, the polyphenol content loss was much lower compared to the previous results previous to FOS addition. The mixture of Batch I + galactomannans + FOS showed a 51 % decrease in the TPP after digestion (2.31- 1 .14%), and the same result was obtained for the Batch II + galactomannans + FOS blend (2.17-1 .06%); as average, the decrease was about 50 % (from 2.2 to 1 .1 %; Table 2a).
3. Antioxidant capacity
The extracts and their corresponding mixtures with galactomannans and FOS were assessed for their antioxidant capacity before and after digestion. As shown in Table 2a, the antioxidant capacity followed similar trend to the Total Polar Phenols content as expected due to their direct correlation. For the Batch I carob extract, the antioxidant capacity expressed by Trolox ratio was 12.8%, and after digestion, was reduced by approximately 88% (12.8-1 .6%), and for the Batch II extract, a 71 % (12.8-3.7%); as average, the antioxidant capacity was reduced in the extract about 80 % (from 12.8 to 2.6 %). Once to the extract was added the locust bean gum with GLMN, the extract followed the same trend, reducing its antioxidant capacity as average about 50 % (from 7.2 to 3.8 %). When FOS was added, the antioxidant capacity showed an average reduction of about 70 % (from 5.5 to 1 .6 %). The addition of GLMN and FOS to the final composition reduced the effect of the digestion process on the antioxidant capacity between 30 and 10 % respectively compared to the initial extract.
Example 3. Physiological effects of the composition.
In order to demonstrate the increase in the bioaccesibility of the bioactive compound included in the composition of the invention, an in vivo test was carried out. Monitoring the possible beneficial effects of said bioactive compound was used to demonstrate that the absorption is higher and therefore, the levels of sugars and lipids in blood were improved, despite of the consumption of a high fat/high sucrose diet. Experimental design:
Three groups of C57BL/6J 16-week old male mice were used:
- Control mice (n = 12): Fed ad libitum with a standard chow
- HFD mice (n = 12): Fed ad libitum with a high fat/high sucrose diet (Research Diets # D 12331 ) for 12 weeks
- HFD + Composition mice (n = 12): Fed ad libitum with a high fat/high sucrose diet (Research Diets # D12331 ) supplemented with the composition of the invention (4.8%) for 12 weeks. Said composition was obtained according to the method of Example 1 and comprises 36% wt. of GLMN, 3% wt. of FOS and 1 % wt. of the phenolic extract from carob.
Twelve weeks after the beginning of the treatment all mice were fasted for 12 h. at night and the next morning the fasting blood glucose was measured. Subsequently blood was taken from the submaxillary sinus to analyse the plasma levels of insulin, triglycerides, total lipids, total cholesterol, LDL-cholesterol and HDL-cholesterol. As it is shown in Figure 4, mice fed the high fat/high sucrose diet gained more weight during the 12-week period compared to controls (P<0.001 ), regardless of the treatment with the composition of the invention. Thus, the consumption of said composition together with a high fat/high sucrose diet for 12 weeks is not able to reduce body weight gain.
Nevertheless, glucose plasma levels were significantly up regulated in HFD mice compared to controls (P<0.01 ). In these mice, glycaemia was above 120 mg/ml (135 ± 9) which means that after 12 weeks fed with the high fat/high sucrose diet they have become diabetic. On the contrary, glycemia of mice fed the HFD+composition of the invention was significantly reduced compared to HFD mice (109 ± 7; P<0.05). Indeed, these values were similar to those control animals (104 ± 8). These results suggest that supplementation with the composition of the invention for 12 weeks prevents the development of diabetes induced by the consumption of a high fat/high sucrose diet.
Regarding the lipid profile, HFD mice showed increased plasma levels of triglycerides (PO.01 ; Figure 3A), total lipids (PO.01 ; Figure 3B), total cholesterol (PO.001 ; Figure 3C), c-LDL (PO.01 ; Figure 3D) and c-HDL (PO.05; Figure 3E), compared to controls. Treatment with the composition of the invention for 12 weeks did not have an impact neither in the plasma triglycerides, nor in total lipids. However, it significantly reduced the concentrations of total cholesterol (P<0.05) and c-LDL (p<0.05) in plasma and increased c-HDL (P<0.05).
In order to assess endothelial function, quantification of reactive hyperemia was performed twelve weeks after the initiation of the treatment. For that purpose, mice were anesthetized with a continuous flow of isoflurane in 100% oxygen. Cutaneous blood perfusion was recorded using a Laser Speckle Contrast imaging system and the leg blood perfusion after 3 minutes occlusion through tourniquet was studied. The repayment/debt ratio was the ratio of the AUC (Area Under Curve) of the post- occlusive hyperemia (H) and the AUC of perfusion debt (I) during vascular occlusion, showed at Figure 7. All data are represented as mean ± SEM. Area Under Curve data and Statistical analysis was carried out by Origin statistical software (Northampton, Massachusetts, USA). Differences were considered significant when p-value < 0.05. It can be seen at Figure 8 how after 12 weeks of treatment HFHS mice showed decreased hyperemic response compared to controls (p-value < 0.005), being this effect attenuated by supplementation with the composition of the invention in diet (p- value < 0.05).

Claims

1. A composition comprising:
galactomannan in an amount of from 36 to 40% by weight based on the total weight of the composition,
fructooligossacharides in an amount of from 2 to 4% by weight based on the total weight of the composition, and
at least one extract of polar nature.
2. Composition according to claim 1 , wherein the amount of galactomannan is 36% by weight based on the total weight of the composition and/or the amount of fructooligosascharides is 3% by weight based on the total weight of the composition.
3. Composition according to claim 1 or 2, wherein galactomannan comprise a mannose:galactose ratio of 3:1.
4. Composition according to any one of claims 1 to 3, wherein the fructooligossacharides comprise no less than 90% by weight of oligofructose (%, dry matter).
5. Composition according to any one of claims 1 to 4, wherein the extract of polar nature comprises hydrolysable tannins derived from gallic acid.
6. Composition according to claim 5, wherein the hydrolysable tannins derived from gallic acid represent between 50 to 90% by weight of the extract of polar nature, preferably 70 % by weight.
7. Composition according to any one of claims 1 to 6, wherein the extract of polar nature is derived from carob, preferably from carob pods.
8. Composition according to any one of claims 1 to 7, wherein the extract of polar nature is in an amount of from 30 to 49% by weight based on the total weight of the composition, preferably 40% by weight.
9. Composition according to any one of claims 1 to 8 for use as a medicament.
10. Composition according to any one of claims 1 to 8 for use in the treatment and/or prevention of a pathological condition associated to the metabolic syndrome.
1 1. Composition for use according to claim 10 wherein the pathological condition is selected from obesity, endothelial dysfunction, dyslipidaemia, hypertension and hyperglycaemia.
12. A composition for use in increasing the absorption of an extract of polar nature in a subject with the following composition:
galactomannans in an amount of from 36 to 40% by weight based on the total weight of the composition,
fructooligossacharides in an amount of from 2 to 4% by weight based on the total weight of the composition, and
said extract of polar nature.
13. Composition for use according to claim 12, wherein the amount of galactomanans is 36% by weight based on the total weight of the composition and/or the amount of fructooligossacharides is 3% by weight based on the total weight of the composition.
14. Composition for use according to claim 12 or 13, wherein the galactomannans comprise a mannose:galactose ratio of 3:1.
15. Composition for use according to any one of claims 12 to 14, wherein the fructooligossacharides comprise no less than 90% by weight of oligofructose (%, dry matter).
16. Composition for use according to any one of claims 12 to 15, wherein the extract of polar nature comprises hydrolysable tannins derived from gallic acid.
17. Composition for use according to claim 16, wherein the hydrolysable tannins derived from gallic acid represent between 50 to 90% by weight of the extract of polar nature, preferably 70% by weight.
18. Composition for use according to any one of claims 12 to 17, wherein the extract of polar nature is derived from carob, preferably from carob pods.
19. Composition for use according to any one of claims 12 to 18, wherein the extract of polar nature is in an amount of from 30 to 49% by weight based on the total weight of the composition, preferably 40% by weight.
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